There are two major long range goals for the research carried out in this laboratory. The first of these is to fully characterize the terminal three membrane associated enzymes of the heme biosynthetic pathway. This characterization includes both basic biochemical studies and examination of the mechanism and regulation of their biosynthesis and translocation to the inner mitochondrial membrane. The second major goal is to characterize regulatory mechanisms that exist in the heme biosynthetic pathway in both erythroid and nonerythroid cell types. It is hoped that by defining the nature of the events that occur in normal cells, it will be possible to get a better understanding of why individuals with genetic lesions in the heme biosynthetic machinery (porphyrias) show variable penetrance of the traits. The specific experiments in this proposal are designed to approach these two general goals via several different avenues. These can be broadly grouped into four categories: 1) continued characterization of the regulation of the temporal synthesis of the terminal three enzymes during erythroid cell differentiation, 2) examination of the proposed cellular regulatory heme pool, 3) biochemical and molecular examination of ALA synthase, and 4) examination of the regulation and roles of the two ALA synthases in erythroid and nonerythroid cells. In all of these experiments cultured murine erythroleukemia (MEL) cells induced to differentiate by DMSO are used as an erythroid cell model, while cultured mouse hepatoma cells (Hepa 1c1c7) are employed as a non-erythroid cell model. The data obtained form these studies will serve as a foundation for researchers in the clinical arena whose concerns are the medical disorders of heme biosynthesis (the porphyrias and anemias) and red cell biogenesis (leukemias). In addition these studies should be of value to toxicologists who target substances have an affect on cellular heme metabolism.